HORMONES
AND
BEHAVIOR
10,
The Estrogenic
11% 127 (1978)
Arousal of Aggressive Female Mice1
Behavior
in
NEALG.SIMONANDRONALDGANDELMAN* Department
qf Psychology,
Rutgers
University,
AJew Brutlswick,
Nebv Jersey 08903
Experiments were conducted to determine the conditions under which estrogen would promote male-like aggressive behavior in female mice. The results of the first experiment showed that most females chronically exposed to testosterone propionate (TP) in adulthood fought, whereas females similarly treated with estradiol benzoate (EB) did not display aggression. Another experiment found that, when either TP or EB was administered on the day of birth, adult females displayed aggression in response to daily EB injections during adult life. Also, the potentiating effect of neonatal hormone exposure declined over the first 12 days postpartum, as 100% of the Day 0.75% of the Day 6. and 0% of the Day 12 and 18 TP-treated females fought in response to daily injections of 40 Erg of EB in adulthood. The final study showed that, under the test conditions employed, the failure of a chronic adult EB regimen to promote aggression was not due to a competing tendency to display female sexual behavior.
Evidence suggests that estrogen (E) produces effects upon physiology and behavior which resemble those produced by testosterone (T). Either hormone, when administered to the neonatal female rat, produces irregular ovarian cyclicity and anovulatory sterility (cf. Doughty, Booth, McDonald, and Parrott, 1975; Harris and Levine, 1965; Whalen, Luttge, and Gorzalka, 1971; Whalen and Nadler, 1963). Further, exposure of neonatal female mice or rats to testosterone or estrogen potentiates later response to the aggression- and male sex behavior-activating properties of testosterone (Barr, Gibbons, and Moyer, 1977; Bronson and Desjardins, 1970; Edwards, 1969; Edwards and Herndon, 1970; Whitsett, Bronson, Peters, and Hamilton, 1972). This similarity between estrogen and testosterone extends to manipulations made in adulthood, since both copulatory and aggressive behaviors can be restored in castrate adult male mice and rats by the administration of either steroid (Beeman, 1947; Edwards and ’ This research was supported by Grant MH-28660 from NICHD, NIH, and by NSF Grant BNS-07347. ? To whom requests for reprints should be addressed at: Department of Psychology, Busch Campus, Rutgers University, New Brunswick, New Jersey 08903.
118 0018-506X/78/0102-0118$01.00/0 Copyright AU rights
@ 1978 by Academic Press, Inc. of reproduction in any form reserved.
ESTROGEN
AND AGGRESSION
IN MICE
119
Burge, 1971; Finney and Erpino, 1976; Luttge, 1972; Sodersten, 1973; Wallis and Luttge, 1975). The following series of experiments was designed to explore further the commonality between estrogen and testosterone by studying the relationship between estrogen and aggression, a behavior generally exhibited by males but infrequently shown by nonlactating females. Specifically, the experiments delineate those conditions under which estrogen can promote fighting in adult female mice. EXPERIMENT
1
Adult ovariectomized female mice given a chronic testosterone propionate (TP) regimen can be induced to display aggressive behavior toward a stimulus male (Barkley and Goldman, 1977a,b; Svare, Davis, and Gandelman, 1974). Since both estradiol benzoate (EB) and TP restore aggression in male mice castrated as adults (Edwards and Burge, 1971; Finney and Erpino, 1976), it is possible that a chronic regimen of EB given to adult, ovariectomized females also will induce aggression. The following experiment assesses this possibility. Method
Seventy-five female Rockland-Swiss (R-S) albino mice, maintained as an outbred strain in a closed colony, were used. They were provided with food and water in excess and were kept on a 12-hr light/dark cycle wit lights on at 0700 hr. The females were weaned on Day 21 and remained housed five or six per cage with littermates until Day 65 of life, when they were ovariectomized under ether anesthesia and housed singly in 28 x 8 x 13-cm cages. Following a 3-day recovery period, the females were assigned randomly to one of five treatment groups of 15 animals each, These animals received daily subcutaneous injections of either 0.5, 10,20: or 40 pg of EB or 500 ,ug of TP throughout the experiment. The hormones were dissolved in 0.02 ml of sesame oil and were administered between 1600 and 1800 hr. The initial aggression test occurred the morning following the second hormone injection. Tests: conducted between 0900 and 1100 hr, consisted of leaving an olfactory bulbectomized male in the home cage of the test animal for 10 min. Bulbectomized males were used as opponents because, while neither initiating nor responding to attack by fighting back, they reliably elicit aggression comparable to that of intact mates (Denenberg, Gaulin-Kremer, Gandelman, and Zarrow, 1973). Thus, any aggression seen is both initiated and terminated by the test animal. The females were tested every other day for 50 days (25 testsj or until aggression was observed. The criterion for aggression was at least 5 set of persistent biting and chasing of the stimulus male. Measures recorded were the latency in days for aggression to appear, total duration of fighting, and
120
SIMON
AND GANDELMAN
number of attacks. An attack was considered terminated when the test animal turned away from the stimulus male. Finally, an attack ratio score, which, we feel, reflects the intensity of aggression, was calculated by dividing total attack duration by number of attacks. Results
With the exception of one female from the 40-pg group, no fighting was displayed by any of the EB-treated females. In contrast, 12/H TPexposed females displayed aggressive behavior. Obviously, TP was effective and EB was ineffective in promoting aggression in adult female mice. EXPERIMENT
2
This experiment asks whether the ineffectiveness of EB in promoting aggression in adult females was due to the manner by which the hormone was administered rather than to an inability of the steriod to produce aggression. This possibility was tested by administering estrogen in a Silastic capsule, which releases its contents slowly and relatively constantly, thus eliminating the rather large variations in estrogen levels resulting from subcutaneous injection. Also, it has been reported that estrogen-containing Silastic capsules have been used successfully to induce male sex behavior in adult, ovariectomized rats (Emery and Sachs, 1975). Method
Twenty 65-day-old females, reared, housed, and gonadectomized as described in Experiment 1, were used. Following a 3-day recovery period, they were assigned randomly to one of two groups. One group of animals was implanted with a IO-mm length of Silastic tubing containing 5 mg of estradiol suspended in 0.02 ml of oil, whereas the other group was implanted with an oil-filled capsule. The capsules were placed under the skin of the neck while the animals were anesthetized with ether. Aggression testing commenced 48 hr after implantation. The testing procedure and measures recorded were as previously described. Results
No fighting was exhibited by either estrogen- or oil-implanted animals. Thus, the manner in which EB was administered in the first experiment apparently did not contribute to the inability of estrogen to induce aggressive behavior in the adult female. EXPERIMENT
3
The first two experiments showed that a chronic EB regimen given to ovariectomized females could not produce aggressive behavior. Since the neonatal administration of either EB or TP to female mice facilitates later
ESTROGEN
AND AGGRESSION
IN MICE
t21
responding to the aggression-promoting property of T (Bronson and Desjardins, 1970; Edwards, 1969; Edwards and Herndon, 1970; Whitsett el al., 1972), it is possible that such treatment would potentiate the ability of EB to promote aggression when given in adulthood. The following experiment investigated this possibility by exposing females to either EB, TP, or oil on the day of birth and then testing them for aggression during exposure to EB in adulthood. Seventy-eight l-day-old female R-S mice were selected at random and given, in groups of five or six, to foster mothers that had delivered within the previous 24 hr and whose own young had been removed. The 1.5foster litters thus formed were divided into three groups of five litters. The young of one group of litters were injected subcutaneously with 300 pg of TP, those of a second group, with 50 pg of EB, and those of a third group with the oil vehicle. The hormones were dissolved in 0.02 ml of sesame oil and were administered within 18 hr of birth. The animals were weaned on Day 21 and kept in littermate groups until Day 65, when they were ovariectomized and housed singly, Three days after ovariectomy, the females were assigned randomly to one of three adult treatment groups: either 40 pg of EB, 0.5 pg of EB, or oil. Thus, nine groups of animals, treated in infancy and in adulthood, were formed. They were: TP-oil (n = 4) (TP in infancy followed by injections of oil in adulthood); TP-0.5 pg of EB (n = 11); TP-40 pg of EB (n = Ii); EB-oiE (n = 4); EB-0.5 pg of EB (12= 11); EB-40 pg of EB (n = i 1); oil-oil (n = 4): oil-O.5 pg of EB (n = 11); and oil-40 pg of EB (n = 11). Subcutaneous injections were given daily throughout the course of the experiment. The initial aggression test was given the morning following the second injection. The testing procedure and measures recorded were as described previously. A summary of the data is given in Table 1. Twenty-nine of forty-four mice that received steroids both neonatally and in adulthood fought, whereas O/34 given oil at either time displayed aggression. In order to compare the effectiveness of neonatal EB and TP administration, data were collapsed across the two adult EB doses. This manipulation showed that the two neonatal treatments were equally effective in potentiating fighting, as 1§/22neonatally TP-exposed and 14/22neonatally EB-exposed females fought during adult EB administration. To determine if the proportion of females fighting differed due to adult treatment, the data were collapsed across the neonatal TP and EB conditions. Twelve of twenty-~ IWOfemales fought when administered 0.S pg of EB in adulthood, while 17/22 displayed aggression in response to the 40 pg of EB. These proportions are not significantly different.
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The mean latency in days for aggression to appear in females that fought was 7.2 for EB-0.5 pg of EB, 7.1 for EB-40 pg of EB, 11.3 for TP-0.5 ,zg of EB, and Il. 1 for TP-40 ,ug of EB. An analysis of variance revealed that groups did not differ significantly on this measure. Similarly, an analysis showed that groups did not differ significantly on the attack ratio score. Group means ranged from 1.5 for the TP-0.5 pg of EB animals, to point 1 for both the TP-40 pg of EB and EB-40 pg of EB females. Thus, the data show that hormones must be administered neonatally for aggression to appear during the administration of EB in adult life. Both neonatal TP and EB were equally effective in potentiating the ability of estrogen to induce aggression, and a dose of 0.5 pg of EB was sufficient to promote fighting in more than 50% of the females. EXPERIMENT
4
The results of Experiment 3 demonstrated that gonadal hormones must be present neonatally for EB to produce aggression in adult female mice. In light of this finding, it was of interest to delineate the period within which TP could be administered such that adult EB exposure would produce aggression. This question was addressed by administering TP to separate groups of animals on different days during early development and then testing for aggression during exposure to EB in adult life. The 40-pg dose of EB was chosen for the adult regimen because, although not TABLE I The Proportion Fighting, Latency for Aggression to Appear, and Attack Ratio Scores of Female Mice Administered TP, EB, or Oil on the Day of Birth and EB or Oil during Adulthood0 Group Neonatal TP TP EB EB Oil Oil TP EB Oil
Adult 0.5 pg of EB 40 pg of EB 0.5 fig of EB 40 pg of EB 0.5 c(g of EB 40 pg of EB Oil Oil Oil
Proportion fightingb 6/11 9111 6/11 S/II O/l1 O/II 014 o/4 014
(55%) (83%) (55%) (73%) ( 0%) ( 0%) ( 0%) ( 0%) ( 0%)
Latencyc (days)
Attack ratioC
II.5 11.1 7.2 7.1 -d -
I.5 2.1 2.0 2.1 -d -
a Latency and attack ratio means were calculated using data only from animals which fought. a Significant differences among groups. c No significant differences. d No fighting was observed.
ESTROGEN
AND AGGRESSION
123
IN MICE
significantiy different from the OS-pg EB dose, it was somewhat more effective in promoting aggression following steroid injections on the day of birth. Seventy-two randomly selected R-S females, housed six per cage with a foster mother whose own young had been removed and reared as previously described, were used. The litters were assigned to one of eight groups which differed with respect to the day of treatment and the hormone administered. Treatments, given on Day 0. 6, 12, or 18 of life, consisted of a subcutaneous injection between 0800 and 1000 hr of either 300 pg of TP in 0.02 ml of oil or an equal volume of the oil vehicle. On each of the four injection days, two litters received TP and one was given oil. The animals were ovariectomized and housed singly on Day 65. Following a 3-day recovery period. all females began receiving daily subcutaneous injections of 40 pg of EB in 0.02 ml of oil between 1600and 1800 hr. The injections were continued throughout the study. The initial aggression test occurred on the morning following the second injection The testing procedure and the measures recorded were as previously described. The data are summarized in Table 2. None of the females receiving oil on any of the 4 days during early life or TP on Day 12 or 18 fought upon exposure to EB in adulthood. In contrast, 100% of the Day 0 and 75% of the Day 6 TP-treated females fought in response to EB exposure in TABLE 2 The Proportion Fighting, Latency for Aggression to Appear, and Attack Ratio Scores of Female Mice Administered TP or Oil on Day 0, 6, 12, or 18 of Life and 40 pg EB during Adulthood” Day administered TP TP TP TP Oil Oil Oil Oil
0 6 12 18 0 6 12 18
Proportion 12112 9112 O/l? 0112 O/6 016 016 016
fightingb
Latency6 (days)
Attack ratio’
3.1 i4.0 -d -
2.3 2.3 -d -
(loo%) ( 75%) ( 0%) ( 0%) ( 0%) ( 0%) ( 0%) ( 0%)
Ii The latency and attack ratio means were calculated fought. 3 Significant differences among groups. c No significant differences. d No fighting was observed.
-
using data only from females which
124
SIMON
AND GANDELMAN
adulthood. This difference in the percentage of animals given TP on Days 0 and 6 that fought was statistically significant (Fisher’s exact probability test, P < 0.01). In addition, t tests revealed that the Day 0 TP-treated animals fought sooner following the commencement of adult EB treatment (x = 3.1 days) than did mice exposed to TP on Day 6 (x = 14.0 days) [t( 19) = 3.88, P < 0.0051.A final t test showed that these two groups did not differ on the attack ratio measure, both having means of 2.3. The data, then, show that the administration of EB during adulthood produces fighting behavior only in animals exposed to TP on Day 6 of life and earlier but not on Day 12. (It is possible, albeit unlikely, that exposure to TP on Day 11 also would have potentiated the estrogenic activation of aggression.) Also, the earlier the female is exposed to TP, the more effective the EB treatment will be in promoting aggression. EXPERIMENT
5
Chronic exposure of the adult female mouse to estrogen will induce sexual receptivity (Luttge, Jasper, Gray, and Sheets, 1977). Thus, it may be that animals given EB in adulthood in the absence of neonatal exposure to TP or EB did not fight because the adult regimen promoted a tendency to display female sex behavior, a response perhaps incompatible with fighting. Such an outcome could not have been observed in the previous experiments because the olfactory bulbectomized males which were used as stimulus animals do not display sex behavior, presumably because they cannot perceive the pheromones released by females. In order to determine whether the chronic adult EB treatment did induce sex behavior, adult females were administered TP, EB, or oil and tested for sexual receptivity with intact males. Method Thirty-six randomly selected R-S females. reared and maintained under conditions previously described, were used. They were ovariectomized and housed individually on Day 65. On Day 68, the females were assigned to one of six groups of six animals each, based on the hormone administered and the duration of the treatment period. The treatments were daily subcutaneous injections of either 0.5 pg of EB, 40 pg of EB, 500 ,ug of TP, or oil vehicle for either 20 or 28 days. These treatment periods were chosen because one terminates before, and the other after, the average number of days required for TP to induce aggression in adult female mice (22 days, Svare et al., 1974). Tests for female sex behavior were given on treatment Days 16 and 20 for the short-exposure groups and on Days 24 and 28 for the long-exposure animals. The testing procedure, conducted 3 to 5 hr into the dark portion of the light/dark cycle under dim red illumination consisted of placing the female into a 5-gal terrarium with a sexually experienced male which had been habituated to the enclosure. The test
ESTROGEN
AND AGGRESSION
IN MICE
125
continued for 30 min or until 10 mounts were observed. The number of lordotic responses was recorded. Sex behavior was not displayed by any of the females. Many of the TP-exposed females either fought with or were attacked by the stimulus males. In contrast, the EB- and oil-treated females made no contact with the males. DISCUSSION
The experiments have clearly demonstrated that estrogen can promote aggression in adult female mice only if there is exposure to estrogen or testosterone early in life. Further, under the conditions of this experiment, the absence of fighting in females given EB solely in adulthood does not appear to be due to a competing tendency to display lordotic behavior. This result is surprising in light of a report demonstrating that a lo-day EB regimen would induce female sexual behavior in ovariectomized, adult mice (Luttge et ai., 1977). However, differences in both doses of EB employed and treatment duration prior to testing may underly this discrepancy. The data, then, suggest that the presence or absence of gonadal steroids during early development determined the subsequent behavioral response to estrogen in adulthood; if present, the response was male-like (fighting), if absent, female-like (passivity). The data indicate, therefore, that passivity of the nonlactating female mouse during interactions with adult males may depend upon relative ovarian quiesence during early postnatal life, a situation which does occur normally (Dohler and Wuttke, 19X), The possibility that a relatively quiescent ovary is necessary during early life for normal female development has been raised by Gorski (1963) and, in addition to the current findings, is supported by studies demonstrating that neonatal estrogen administration suppresses. lordotic responding in castrate females primed with estrogen/progesterone in adulthood (Whalen and Nadler, 1963) while increasing the display of male sex behavior by intact female rats (Dorner, Docke, and Hinz, 1971). The results of the present study also bear on the hypothesis that T exerts its effects upon the central nervous system after being aromatized to estrogen (Naftolin, Ryan, and Petro, 1971; Ryan, Naftolin, Reddy, Flores, and Petro, 1974). This view draws support, in part, from studies showing that estrogen is recovered as a metabolite of testosterone from hypothalamic nuclei of neonatal and adult male rats (Leiberburg and McEwen, 1975a,b). Further, estrogen, like testosterone, will restore the sexual and aggressive behavior of castrate adult males (Beeman, 1947; Edwards, 1969; Edwards and Burge, 1971; Finney and Erpino, 1976; Luttge, 1972; Sodersten, 1973; Wallis and Luttge, 1975). The findings of
126
SIMON AND GANDELMAN
the present experiments, however, do not support the aromatization hypothesis, since EB, unlike TP, was incapable of activating malelike aggressive behavior in the adult, non-neonatally steroid exposed female. How might this discrepancy be explained? A common feature of previous work showing that estrogen can restore masculine behaviors in adult rodents is that gonadal hormones were present neonatally (cf. Finney and Erpino, 1976; Sodersten, 1973). In addition, it has been proposed that males can aromatize T to E and utilize it for the promotion of behavior only as a consequence of neonatal exposure to gonadal steroids (Ohno, Geller, and YoungLai, 1974). Since estrogen was capable of activating male-like aggression in female mice only if such exposure occurred, the current experiments support this hypothesis. Therefore, in assessing the role of aromatization in the promotion of male-like behavior, there is a need to study the effects of hormones upon animals that have not been exposed to gonadal steroids early in life. REFERENCES Barkley, M. S., and Goldman, B. D. (1977a). Testosterone-induced aggression in adult female mice. Horm. Behav. 9, 76-84. Barkley, M. S., and Goldman, B. D. (1977b). The effects of castration and Silastic implants of testosterone on intermale aggression in the mouse. Horm. Behav. 9, 32-48. Barr, G. A., Gibbons, L. J., and Moyer, K. D. (1977). Male-female differences and the influence of neonatal and adult testosterone on intraspecies aggression in rats. J. Camp. Physiol. Psychol. 90, 1169-I 183. Beeman, E. A. (1947). The effect of male hormone on aggressive behavior in mice. Physiol. Zool. 20, 373-40s. Bronson, F. H., and Desjardins, C. (1970). Neonatal androgen administration and adult aggressiveness in female mice. Gen. Comp. Etzdocrinol. 15, 320-325. Denenberg, V. H., Gaulin-Kremer, E., Gandelman, R., and Zarrow, M. X. (1973). The development of standard stimulus animals for mouse (Mus musculus) aggression testing by means of olfactory bulbectomy. Anim. Behav. 21, 590-598. Dohler, K. D., and Wuttke, W. (1975). Changes with age in levels of serum gonadotrophins, 97, prolactin, and gonadal steroids in prepubertal male and female rats. Endocrinology 898-907. Dorner, G., Docke, F., and Hinz, G. (1971). Paradoxical effects of estrogen on brain differentiation. Neuroendocrinology 7, 146-1.55. Doughty, C., Booth, J. E., McDonald, P. G., and Parrott, R. F. (1975). Effects of oestradiol-17p, oestradiol benzoate, and the synthetic oestrogen RU-2858 on sexual differentiation in the neonatal female rat. J. Endocrinol. 67, 419-424. Edwards, D. A. (1969). Early androgen stimulation and aggressive behavior in male and female mice. Physiol. Behnls. 4, 333-338. Edwards. D. A., and Burge, K. G. (1971). Estrogenic arousal of aggressive behavior and masculine sexual behavior in male and female mice. Horm. Bekav. 2, 239-245. Edwards, D. A., and Herndon, J. (1970). Neonatal estrogen stimulation and aggressive behavior in female mice. Physiol. Behav. 5, 993-995. Emery, D. E., and Sachs, B. D. (1975). Ejaculatory pattern in female rats without androgen treatment. Scierzce 190, 484-486. Finney, H. C., and Erpino, M. J. (1976). Synergistic effect of estradiol benzoate and dihydrotestosterone on aggression in mice. Horm. Behav. 7, 391-400.
ESTROGEN AND AGGRESSION IN MICE
12:
Gorski. R. A. (1963). Modification of ovulatory mechanisms by postnatal administration of estrogen to the rat. Amer. J. Physiol., 205, 842-844. Harris, C. W., and Levine, S. (1965). Sexual differentiation of the brain and its experimenta control. J. Phq’siol. 181, 379-400. Leiberburg, I.. and McEwen, B. S. (1975a). Estradiol 17-p: A metabolite of testosterone recovered in cell nuclei from limbic areas of neonatal rat brains. Brai Res. 85, 165-170. Leiberbog, I.. and McEwen, B. S. (1975b). Estradiol 17-p: .4 metaboiite of testosterone recovered in cell nuclei from limbic areas of adult male rat brains Bruin Res. 85, 171-174. Luttge, W. G. (1972). Activation and inhibition of isolation induced intermale fighting behavior in castrate male CD-I mice treated with steroidal hormones. Harm. Eehal~. 3, 71-81. Luttage, W. G., Jasper. T. W., Gray,H. E.,andSheets, C. S. (1977). Estrogen-inducedsexuai receptivity and localization of “H-estradiol in brains of female mice: Effects of 5areduced androgens, progestins, and cyproterone acetate. Pharnracol Biocizem. Bsha~l. 6, 521-5?8. Naftolin, F., Ryan, K. J., and Petro, Z. (1971). Aromatization of androstenedione by the anterior hypothalamus of adult male and female rats. Endocrinology 90, 295-297. Ohno, S., Geller, L. N., and YoungLai, E. V. (1974). Tfm mutation and masculinization versus feminization of the mouse central nervous system. Cell 3, 235-242. Ryan, K. J,, Naftolin, F., Reddy, V., Flares, F., and Petro, Z. (1974). Estrogen formation in the brain. Amer. J. Obstet. Gwecol. 114, 454-460. Sodersten, P. (1973). Estrogen-activated sexual behavior in male rats. Harm. Beha:,. 4, 247-256. Svare, B.. Davis, P. G., and Gandelman, R. (1974). Fighting behavior io female mice following chronic androgen treatment during adulthood. PIz>lsiol. Beiza~~.12, 399-403, Wailis, C. J., and Luttge, W. G. (1975). Maintenance of male sexual behaviour by combined treatment with oestrogen and dihydrotestosterone in CD-1 mice. J. Endociinol. 66, 257-262. Whalen. R. E., Luttge, W. G., and Gorzaika, B. B. (1971). Neonatal androgenization and the development of estrogen responsivity in male and female rats. Norr?2. Behars. 2, 83-90. Whalen, R. E., and Nadler, R. D. (1963). Suppression of the devetopment of female mating behavior by estrogen administered in infancy. Science 140, 273-274. Whitsett, J. M., Bronson, F. H., Peters, P. J., and Hamilton, T. H. (1972). Neonatal organization of aggression in mice: Correlation of critical period with uptake or hormone. Harm. Behav. 3, 11-21.
AND
BEHAVIOR
10,
The Estrogenic
11% 127 (1978)
Arousal of Aggressive Female Mice1
Behavior
in
NEALG.SIMONANDRONALDGANDELMAN* Department
qf Psychology,
Rutgers
University,
AJew Brutlswick,
Nebv Jersey 08903
Experiments were conducted to determine the conditions under which estrogen would promote male-like aggressive behavior in female mice. The results of the first experiment showed that most females chronically exposed to testosterone propionate (TP) in adulthood fought, whereas females similarly treated with estradiol benzoate (EB) did not display aggression. Another experiment found that, when either TP or EB was administered on the day of birth, adult females displayed aggression in response to daily EB injections during adult life. Also, the potentiating effect of neonatal hormone exposure declined over the first 12 days postpartum, as 100% of the Day 0.75% of the Day 6. and 0% of the Day 12 and 18 TP-treated females fought in response to daily injections of 40 Erg of EB in adulthood. The final study showed that, under the test conditions employed, the failure of a chronic adult EB regimen to promote aggression was not due to a competing tendency to display female sexual behavior.
Evidence suggests that estrogen (E) produces effects upon physiology and behavior which resemble those produced by testosterone (T). Either hormone, when administered to the neonatal female rat, produces irregular ovarian cyclicity and anovulatory sterility (cf. Doughty, Booth, McDonald, and Parrott, 1975; Harris and Levine, 1965; Whalen, Luttge, and Gorzalka, 1971; Whalen and Nadler, 1963). Further, exposure of neonatal female mice or rats to testosterone or estrogen potentiates later response to the aggression- and male sex behavior-activating properties of testosterone (Barr, Gibbons, and Moyer, 1977; Bronson and Desjardins, 1970; Edwards, 1969; Edwards and Herndon, 1970; Whitsett, Bronson, Peters, and Hamilton, 1972). This similarity between estrogen and testosterone extends to manipulations made in adulthood, since both copulatory and aggressive behaviors can be restored in castrate adult male mice and rats by the administration of either steroid (Beeman, 1947; Edwards and ’ This research was supported by Grant MH-28660 from NICHD, NIH, and by NSF Grant BNS-07347. ? To whom requests for reprints should be addressed at: Department of Psychology, Busch Campus, Rutgers University, New Brunswick, New Jersey 08903.
118 0018-506X/78/0102-0118$01.00/0 Copyright AU rights
@ 1978 by Academic Press, Inc. of reproduction in any form reserved.
ESTROGEN
AND AGGRESSION
IN MICE
119
Burge, 1971; Finney and Erpino, 1976; Luttge, 1972; Sodersten, 1973; Wallis and Luttge, 1975). The following series of experiments was designed to explore further the commonality between estrogen and testosterone by studying the relationship between estrogen and aggression, a behavior generally exhibited by males but infrequently shown by nonlactating females. Specifically, the experiments delineate those conditions under which estrogen can promote fighting in adult female mice. EXPERIMENT
1
Adult ovariectomized female mice given a chronic testosterone propionate (TP) regimen can be induced to display aggressive behavior toward a stimulus male (Barkley and Goldman, 1977a,b; Svare, Davis, and Gandelman, 1974). Since both estradiol benzoate (EB) and TP restore aggression in male mice castrated as adults (Edwards and Burge, 1971; Finney and Erpino, 1976), it is possible that a chronic regimen of EB given to adult, ovariectomized females also will induce aggression. The following experiment assesses this possibility. Method
Seventy-five female Rockland-Swiss (R-S) albino mice, maintained as an outbred strain in a closed colony, were used. They were provided with food and water in excess and were kept on a 12-hr light/dark cycle wit lights on at 0700 hr. The females were weaned on Day 21 and remained housed five or six per cage with littermates until Day 65 of life, when they were ovariectomized under ether anesthesia and housed singly in 28 x 8 x 13-cm cages. Following a 3-day recovery period, the females were assigned randomly to one of five treatment groups of 15 animals each, These animals received daily subcutaneous injections of either 0.5, 10,20: or 40 pg of EB or 500 ,ug of TP throughout the experiment. The hormones were dissolved in 0.02 ml of sesame oil and were administered between 1600 and 1800 hr. The initial aggression test occurred the morning following the second hormone injection. Tests: conducted between 0900 and 1100 hr, consisted of leaving an olfactory bulbectomized male in the home cage of the test animal for 10 min. Bulbectomized males were used as opponents because, while neither initiating nor responding to attack by fighting back, they reliably elicit aggression comparable to that of intact mates (Denenberg, Gaulin-Kremer, Gandelman, and Zarrow, 1973). Thus, any aggression seen is both initiated and terminated by the test animal. The females were tested every other day for 50 days (25 testsj or until aggression was observed. The criterion for aggression was at least 5 set of persistent biting and chasing of the stimulus male. Measures recorded were the latency in days for aggression to appear, total duration of fighting, and
120
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AND GANDELMAN
number of attacks. An attack was considered terminated when the test animal turned away from the stimulus male. Finally, an attack ratio score, which, we feel, reflects the intensity of aggression, was calculated by dividing total attack duration by number of attacks. Results
With the exception of one female from the 40-pg group, no fighting was displayed by any of the EB-treated females. In contrast, 12/H TPexposed females displayed aggressive behavior. Obviously, TP was effective and EB was ineffective in promoting aggression in adult female mice. EXPERIMENT
2
This experiment asks whether the ineffectiveness of EB in promoting aggression in adult females was due to the manner by which the hormone was administered rather than to an inability of the steriod to produce aggression. This possibility was tested by administering estrogen in a Silastic capsule, which releases its contents slowly and relatively constantly, thus eliminating the rather large variations in estrogen levels resulting from subcutaneous injection. Also, it has been reported that estrogen-containing Silastic capsules have been used successfully to induce male sex behavior in adult, ovariectomized rats (Emery and Sachs, 1975). Method
Twenty 65-day-old females, reared, housed, and gonadectomized as described in Experiment 1, were used. Following a 3-day recovery period, they were assigned randomly to one of two groups. One group of animals was implanted with a IO-mm length of Silastic tubing containing 5 mg of estradiol suspended in 0.02 ml of oil, whereas the other group was implanted with an oil-filled capsule. The capsules were placed under the skin of the neck while the animals were anesthetized with ether. Aggression testing commenced 48 hr after implantation. The testing procedure and measures recorded were as previously described. Results
No fighting was exhibited by either estrogen- or oil-implanted animals. Thus, the manner in which EB was administered in the first experiment apparently did not contribute to the inability of estrogen to induce aggressive behavior in the adult female. EXPERIMENT
3
The first two experiments showed that a chronic EB regimen given to ovariectomized females could not produce aggressive behavior. Since the neonatal administration of either EB or TP to female mice facilitates later
ESTROGEN
AND AGGRESSION
IN MICE
t21
responding to the aggression-promoting property of T (Bronson and Desjardins, 1970; Edwards, 1969; Edwards and Herndon, 1970; Whitsett el al., 1972), it is possible that such treatment would potentiate the ability of EB to promote aggression when given in adulthood. The following experiment investigated this possibility by exposing females to either EB, TP, or oil on the day of birth and then testing them for aggression during exposure to EB in adulthood. Seventy-eight l-day-old female R-S mice were selected at random and given, in groups of five or six, to foster mothers that had delivered within the previous 24 hr and whose own young had been removed. The 1.5foster litters thus formed were divided into three groups of five litters. The young of one group of litters were injected subcutaneously with 300 pg of TP, those of a second group, with 50 pg of EB, and those of a third group with the oil vehicle. The hormones were dissolved in 0.02 ml of sesame oil and were administered within 18 hr of birth. The animals were weaned on Day 21 and kept in littermate groups until Day 65, when they were ovariectomized and housed singly, Three days after ovariectomy, the females were assigned randomly to one of three adult treatment groups: either 40 pg of EB, 0.5 pg of EB, or oil. Thus, nine groups of animals, treated in infancy and in adulthood, were formed. They were: TP-oil (n = 4) (TP in infancy followed by injections of oil in adulthood); TP-0.5 pg of EB (n = 11); TP-40 pg of EB (n = Ii); EB-oiE (n = 4); EB-0.5 pg of EB (12= 11); EB-40 pg of EB (n = i 1); oil-oil (n = 4): oil-O.5 pg of EB (n = 11); and oil-40 pg of EB (n = 11). Subcutaneous injections were given daily throughout the course of the experiment. The initial aggression test was given the morning following the second injection. The testing procedure and measures recorded were as described previously. A summary of the data is given in Table 1. Twenty-nine of forty-four mice that received steroids both neonatally and in adulthood fought, whereas O/34 given oil at either time displayed aggression. In order to compare the effectiveness of neonatal EB and TP administration, data were collapsed across the two adult EB doses. This manipulation showed that the two neonatal treatments were equally effective in potentiating fighting, as 1§/22neonatally TP-exposed and 14/22neonatally EB-exposed females fought during adult EB administration. To determine if the proportion of females fighting differed due to adult treatment, the data were collapsed across the neonatal TP and EB conditions. Twelve of twenty-~ IWOfemales fought when administered 0.S pg of EB in adulthood, while 17/22 displayed aggression in response to the 40 pg of EB. These proportions are not significantly different.
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The mean latency in days for aggression to appear in females that fought was 7.2 for EB-0.5 pg of EB, 7.1 for EB-40 pg of EB, 11.3 for TP-0.5 ,zg of EB, and Il. 1 for TP-40 ,ug of EB. An analysis of variance revealed that groups did not differ significantly on this measure. Similarly, an analysis showed that groups did not differ significantly on the attack ratio score. Group means ranged from 1.5 for the TP-0.5 pg of EB animals, to point 1 for both the TP-40 pg of EB and EB-40 pg of EB females. Thus, the data show that hormones must be administered neonatally for aggression to appear during the administration of EB in adult life. Both neonatal TP and EB were equally effective in potentiating the ability of estrogen to induce aggression, and a dose of 0.5 pg of EB was sufficient to promote fighting in more than 50% of the females. EXPERIMENT
4
The results of Experiment 3 demonstrated that gonadal hormones must be present neonatally for EB to produce aggression in adult female mice. In light of this finding, it was of interest to delineate the period within which TP could be administered such that adult EB exposure would produce aggression. This question was addressed by administering TP to separate groups of animals on different days during early development and then testing for aggression during exposure to EB in adult life. The 40-pg dose of EB was chosen for the adult regimen because, although not TABLE I The Proportion Fighting, Latency for Aggression to Appear, and Attack Ratio Scores of Female Mice Administered TP, EB, or Oil on the Day of Birth and EB or Oil during Adulthood0 Group Neonatal TP TP EB EB Oil Oil TP EB Oil
Adult 0.5 pg of EB 40 pg of EB 0.5 fig of EB 40 pg of EB 0.5 c(g of EB 40 pg of EB Oil Oil Oil
Proportion fightingb 6/11 9111 6/11 S/II O/l1 O/II 014 o/4 014
(55%) (83%) (55%) (73%) ( 0%) ( 0%) ( 0%) ( 0%) ( 0%)
Latencyc (days)
Attack ratioC
II.5 11.1 7.2 7.1 -d -
I.5 2.1 2.0 2.1 -d -
a Latency and attack ratio means were calculated using data only from animals which fought. a Significant differences among groups. c No significant differences. d No fighting was observed.
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significantiy different from the OS-pg EB dose, it was somewhat more effective in promoting aggression following steroid injections on the day of birth. Seventy-two randomly selected R-S females, housed six per cage with a foster mother whose own young had been removed and reared as previously described, were used. The litters were assigned to one of eight groups which differed with respect to the day of treatment and the hormone administered. Treatments, given on Day 0. 6, 12, or 18 of life, consisted of a subcutaneous injection between 0800 and 1000 hr of either 300 pg of TP in 0.02 ml of oil or an equal volume of the oil vehicle. On each of the four injection days, two litters received TP and one was given oil. The animals were ovariectomized and housed singly on Day 65. Following a 3-day recovery period. all females began receiving daily subcutaneous injections of 40 pg of EB in 0.02 ml of oil between 1600and 1800 hr. The injections were continued throughout the study. The initial aggression test occurred on the morning following the second injection The testing procedure and the measures recorded were as previously described. The data are summarized in Table 2. None of the females receiving oil on any of the 4 days during early life or TP on Day 12 or 18 fought upon exposure to EB in adulthood. In contrast, 100% of the Day 0 and 75% of the Day 6 TP-treated females fought in response to EB exposure in TABLE 2 The Proportion Fighting, Latency for Aggression to Appear, and Attack Ratio Scores of Female Mice Administered TP or Oil on Day 0, 6, 12, or 18 of Life and 40 pg EB during Adulthood” Day administered TP TP TP TP Oil Oil Oil Oil
0 6 12 18 0 6 12 18
Proportion 12112 9112 O/l? 0112 O/6 016 016 016
fightingb
Latency6 (days)
Attack ratio’
3.1 i4.0 -d -
2.3 2.3 -d -
(loo%) ( 75%) ( 0%) ( 0%) ( 0%) ( 0%) ( 0%) ( 0%)
Ii The latency and attack ratio means were calculated fought. 3 Significant differences among groups. c No significant differences. d No fighting was observed.
-
using data only from females which
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adulthood. This difference in the percentage of animals given TP on Days 0 and 6 that fought was statistically significant (Fisher’s exact probability test, P < 0.01). In addition, t tests revealed that the Day 0 TP-treated animals fought sooner following the commencement of adult EB treatment (x = 3.1 days) than did mice exposed to TP on Day 6 (x = 14.0 days) [t( 19) = 3.88, P < 0.0051.A final t test showed that these two groups did not differ on the attack ratio measure, both having means of 2.3. The data, then, show that the administration of EB during adulthood produces fighting behavior only in animals exposed to TP on Day 6 of life and earlier but not on Day 12. (It is possible, albeit unlikely, that exposure to TP on Day 11 also would have potentiated the estrogenic activation of aggression.) Also, the earlier the female is exposed to TP, the more effective the EB treatment will be in promoting aggression. EXPERIMENT
5
Chronic exposure of the adult female mouse to estrogen will induce sexual receptivity (Luttge, Jasper, Gray, and Sheets, 1977). Thus, it may be that animals given EB in adulthood in the absence of neonatal exposure to TP or EB did not fight because the adult regimen promoted a tendency to display female sex behavior, a response perhaps incompatible with fighting. Such an outcome could not have been observed in the previous experiments because the olfactory bulbectomized males which were used as stimulus animals do not display sex behavior, presumably because they cannot perceive the pheromones released by females. In order to determine whether the chronic adult EB treatment did induce sex behavior, adult females were administered TP, EB, or oil and tested for sexual receptivity with intact males. Method Thirty-six randomly selected R-S females. reared and maintained under conditions previously described, were used. They were ovariectomized and housed individually on Day 65. On Day 68, the females were assigned to one of six groups of six animals each, based on the hormone administered and the duration of the treatment period. The treatments were daily subcutaneous injections of either 0.5 pg of EB, 40 pg of EB, 500 ,ug of TP, or oil vehicle for either 20 or 28 days. These treatment periods were chosen because one terminates before, and the other after, the average number of days required for TP to induce aggression in adult female mice (22 days, Svare et al., 1974). Tests for female sex behavior were given on treatment Days 16 and 20 for the short-exposure groups and on Days 24 and 28 for the long-exposure animals. The testing procedure, conducted 3 to 5 hr into the dark portion of the light/dark cycle under dim red illumination consisted of placing the female into a 5-gal terrarium with a sexually experienced male which had been habituated to the enclosure. The test
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continued for 30 min or until 10 mounts were observed. The number of lordotic responses was recorded. Sex behavior was not displayed by any of the females. Many of the TP-exposed females either fought with or were attacked by the stimulus males. In contrast, the EB- and oil-treated females made no contact with the males. DISCUSSION
The experiments have clearly demonstrated that estrogen can promote aggression in adult female mice only if there is exposure to estrogen or testosterone early in life. Further, under the conditions of this experiment, the absence of fighting in females given EB solely in adulthood does not appear to be due to a competing tendency to display lordotic behavior. This result is surprising in light of a report demonstrating that a lo-day EB regimen would induce female sexual behavior in ovariectomized, adult mice (Luttge et ai., 1977). However, differences in both doses of EB employed and treatment duration prior to testing may underly this discrepancy. The data, then, suggest that the presence or absence of gonadal steroids during early development determined the subsequent behavioral response to estrogen in adulthood; if present, the response was male-like (fighting), if absent, female-like (passivity). The data indicate, therefore, that passivity of the nonlactating female mouse during interactions with adult males may depend upon relative ovarian quiesence during early postnatal life, a situation which does occur normally (Dohler and Wuttke, 19X), The possibility that a relatively quiescent ovary is necessary during early life for normal female development has been raised by Gorski (1963) and, in addition to the current findings, is supported by studies demonstrating that neonatal estrogen administration suppresses. lordotic responding in castrate females primed with estrogen/progesterone in adulthood (Whalen and Nadler, 1963) while increasing the display of male sex behavior by intact female rats (Dorner, Docke, and Hinz, 1971). The results of the present study also bear on the hypothesis that T exerts its effects upon the central nervous system after being aromatized to estrogen (Naftolin, Ryan, and Petro, 1971; Ryan, Naftolin, Reddy, Flores, and Petro, 1974). This view draws support, in part, from studies showing that estrogen is recovered as a metabolite of testosterone from hypothalamic nuclei of neonatal and adult male rats (Leiberburg and McEwen, 1975a,b). Further, estrogen, like testosterone, will restore the sexual and aggressive behavior of castrate adult males (Beeman, 1947; Edwards, 1969; Edwards and Burge, 1971; Finney and Erpino, 1976; Luttge, 1972; Sodersten, 1973; Wallis and Luttge, 1975). The findings of
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the present experiments, however, do not support the aromatization hypothesis, since EB, unlike TP, was incapable of activating malelike aggressive behavior in the adult, non-neonatally steroid exposed female. How might this discrepancy be explained? A common feature of previous work showing that estrogen can restore masculine behaviors in adult rodents is that gonadal hormones were present neonatally (cf. Finney and Erpino, 1976; Sodersten, 1973). In addition, it has been proposed that males can aromatize T to E and utilize it for the promotion of behavior only as a consequence of neonatal exposure to gonadal steroids (Ohno, Geller, and YoungLai, 1974). Since estrogen was capable of activating male-like aggression in female mice only if such exposure occurred, the current experiments support this hypothesis. Therefore, in assessing the role of aromatization in the promotion of male-like behavior, there is a need to study the effects of hormones upon animals that have not been exposed to gonadal steroids early in life. REFERENCES Barkley, M. S., and Goldman, B. D. (1977a). Testosterone-induced aggression in adult female mice. Horm. Behav. 9, 76-84. Barkley, M. S., and Goldman, B. D. (1977b). The effects of castration and Silastic implants of testosterone on intermale aggression in the mouse. Horm. Behav. 9, 32-48. Barr, G. A., Gibbons, L. J., and Moyer, K. D. (1977). Male-female differences and the influence of neonatal and adult testosterone on intraspecies aggression in rats. J. Camp. Physiol. Psychol. 90, 1169-I 183. Beeman, E. A. (1947). The effect of male hormone on aggressive behavior in mice. Physiol. Zool. 20, 373-40s. Bronson, F. H., and Desjardins, C. (1970). Neonatal androgen administration and adult aggressiveness in female mice. Gen. Comp. Etzdocrinol. 15, 320-325. Denenberg, V. H., Gaulin-Kremer, E., Gandelman, R., and Zarrow, M. X. (1973). The development of standard stimulus animals for mouse (Mus musculus) aggression testing by means of olfactory bulbectomy. Anim. Behav. 21, 590-598. Dohler, K. D., and Wuttke, W. (1975). Changes with age in levels of serum gonadotrophins, 97, prolactin, and gonadal steroids in prepubertal male and female rats. Endocrinology 898-907. Dorner, G., Docke, F., and Hinz, G. (1971). Paradoxical effects of estrogen on brain differentiation. Neuroendocrinology 7, 146-1.55. Doughty, C., Booth, J. E., McDonald, P. G., and Parrott, R. F. (1975). Effects of oestradiol-17p, oestradiol benzoate, and the synthetic oestrogen RU-2858 on sexual differentiation in the neonatal female rat. J. Endocrinol. 67, 419-424. Edwards, D. A. (1969). Early androgen stimulation and aggressive behavior in male and female mice. Physiol. Behnls. 4, 333-338. Edwards. D. A., and Burge, K. G. (1971). Estrogenic arousal of aggressive behavior and masculine sexual behavior in male and female mice. Horm. Bekav. 2, 239-245. Edwards, D. A., and Herndon, J. (1970). Neonatal estrogen stimulation and aggressive behavior in female mice. Physiol. Behav. 5, 993-995. Emery, D. E., and Sachs, B. D. (1975). Ejaculatory pattern in female rats without androgen treatment. Scierzce 190, 484-486. Finney, H. C., and Erpino, M. J. (1976). Synergistic effect of estradiol benzoate and dihydrotestosterone on aggression in mice. Horm. Behav. 7, 391-400.
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Gorski. R. A. (1963). Modification of ovulatory mechanisms by postnatal administration of estrogen to the rat. Amer. J. Physiol., 205, 842-844. Harris, C. W., and Levine, S. (1965). Sexual differentiation of the brain and its experimenta control. J. Phq’siol. 181, 379-400. Leiberburg, I.. and McEwen, B. S. (1975a). Estradiol 17-p: A metabolite of testosterone recovered in cell nuclei from limbic areas of neonatal rat brains. Brai Res. 85, 165-170. Leiberbog, I.. and McEwen, B. S. (1975b). Estradiol 17-p: .4 metaboiite of testosterone recovered in cell nuclei from limbic areas of adult male rat brains Bruin Res. 85, 171-174. Luttge, W. G. (1972). Activation and inhibition of isolation induced intermale fighting behavior in castrate male CD-I mice treated with steroidal hormones. Harm. Eehal~. 3, 71-81. Luttage, W. G., Jasper. T. W., Gray,H. E.,andSheets, C. S. (1977). Estrogen-inducedsexuai receptivity and localization of “H-estradiol in brains of female mice: Effects of 5areduced androgens, progestins, and cyproterone acetate. Pharnracol Biocizem. Bsha~l. 6, 521-5?8. Naftolin, F., Ryan, K. J., and Petro, Z. (1971). Aromatization of androstenedione by the anterior hypothalamus of adult male and female rats. Endocrinology 90, 295-297. Ohno, S., Geller, L. N., and YoungLai, E. V. (1974). Tfm mutation and masculinization versus feminization of the mouse central nervous system. Cell 3, 235-242. Ryan, K. J,, Naftolin, F., Reddy, V., Flares, F., and Petro, Z. (1974). Estrogen formation in the brain. Amer. J. Obstet. Gwecol. 114, 454-460. Sodersten, P. (1973). Estrogen-activated sexual behavior in male rats. Harm. Beha:,. 4, 247-256. Svare, B.. Davis, P. G., and Gandelman, R. (1974). Fighting behavior io female mice following chronic androgen treatment during adulthood. PIz>lsiol. Beiza~~.12, 399-403, Wailis, C. J., and Luttge, W. G. (1975). Maintenance of male sexual behaviour by combined treatment with oestrogen and dihydrotestosterone in CD-1 mice. J. Endociinol. 66, 257-262. Whalen. R. E., Luttge, W. G., and Gorzaika, B. B. (1971). Neonatal androgenization and the development of estrogen responsivity in male and female rats. Norr?2. Behars. 2, 83-90. Whalen, R. E., and Nadler, R. D. (1963). Suppression of the devetopment of female mating behavior by estrogen administered in infancy. Science 140, 273-274. Whitsett, J. M., Bronson, F. H., Peters, P. J., and Hamilton, T. H. (1972). Neonatal organization of aggression in mice: Correlation of critical period with uptake or hormone. Harm. Behav. 3, 11-21.
